1. Field
The present disclosure relates to the field of chemical detection. In particular, a method and apparatus for detecting and quantifying bacterial spores on a surface is disclosed.
2. Description of Related Art
Lanthanide complexes, particularly those of terbium (Tb+3) and europium (Eu+3), exhibit luminescence properties for the detection of aromatic biomolecules. The detection scheme is based on the absorption-energy transfer-emission mechanism, which is triggered by the binding of aromatic ligands to lanthanide complexes under UV excitation. Recent efforts have been focused on the detection of dipicolinic acid (DPA) (2,6-pyridinedicarboxylic acid), which is a unique constituent of bacterial spores present at high concentrations (up to 1 M). Dipicolinic acid is also a commercially available product having the following characteristics: CAS #: 499-83-2, Synonyms: 2,6 Pyridine Dicarboxylic Acid, Molecular Formula: C7H5NO4, Molecular Weight: 167.12, Description: White crystalline powder, Sulphated Ash: 0.3% max, Moisture Content: 0.5% max, Melting Point: 242.0 to 245.0.degree. C., Assay: 99.0% min.
Bacterial spores are generally accepted to be indicator species for validating sterility since they are the most resilient form of life against sterilization regimens (Hindle and Hall, 1999 Analyst, 124, 1599-1604). Sterility testing of surfaces is traditionally performed by either (1) swabbing the surface with a cotton applicator, resuspending the swabbed spores, and plating the spore suspension onto growth media; or (2) using Replicate Organism Detection and Counting (RODAC) growth plates that are pressed against a surface to be analyzed. Each of these two bacterial spore assays requires 3-5 days before results are available.
As mentioned, dipicolinic acid (DPA) is present in high concentrations (about 1 molar or about 15% of by weight) in the core of bacterial spores (Murell, 1969, Bact. Spore 1, 216). In its deprotonated state, DPA is dipicolinate (DP) and is found in a 1:1 complex with Ca2+ inside the spore, as shown in FIG. 1A. For all known life-forms, DPA is unique to bacterial spores and is naturally released into bulk solution upon germination—the process of spore-to-vegetative cell transformation. DP can also be released upon lysis of the bacterial spore. Thus, DPA and/or DP are indicator molecules for the presence of bacterial spores. DPA is a classic inorganic chemistry ligand that binds metal ions with high affinity. As mentioned, DPA takes the form of dipicolinate (DP) in its deprotonated form that binds to Ca2+. DPA binding to terbium ions (or other luminescent lanthanide or transition metal ions) triggers intense green luminescence under UV excitation as shown in FIGS. 1B and 1C. The green luminescence turn-on signal indicates the presence of bacterial spores. The intensity of the luminescence can be correlated to the number of bacterial spores per milliliter.
U.S. Patent Application Publication No. 2003-0138876 for “Method bacterial endospore quantification using lanthanide dipicolinate luminescence” discloses a lanthanide that is combined with a medium to be tested for endospores. Dipicolinic acid released from the endospores binds the lanthanides, which have distinctive emission (i.e., luminescence) spectra, and are detected using photoluminescence. The concentration of spores is determined by preparing a calibration curve that relates emission intensities to spore concentrations for test samples with known spore concentrations. A lanthanide complex is used as the analysis reagent, and is comprised of lanthanide ions bound to multidentate ligands that increase the dipicolinic acid binding constant through a cooperative binding effect with respect to lanthanide chloride. The resulting combined effect of increasing the binding constant and eliminating coordinated water and multiple equilibria increases the sensitivity of the endospore assay by an estimated three to four orders of magnitude over prior art of endospore detection based on lanthanide luminescence.
U.S. Patent Application Publication No. 2004-0014154 for “Methods and apparatus for assays of bacterial spores” discloses a sample of unknown bacterial spores which is added to a test strip. The sample of unknown bacterial spores is drawn to a first sample region on the test strip by capillary action. Species-specific antibodies are bound to the sample when the unknown bacterial spores match the species-specific antibodies, otherwise the sample is left unbound. DPA is released from the bacterial spores in the bound sample. Terbium ions are combined with the DPA to form a Tb-DPA complex. The combined terbium ions and DPA are excited to generate a luminescence characteristic of the combined terbium ions and DPA to detect the bacterial spores. A live/dead assay is performed by a release of the DPA for live spores and a release of DPA for all spores. The detection concentrations are compared to determine the fraction of live spores. Lifetime-gated measurements of bacterial spores to eliminate any fluorescence background from organic chromophores comprise labeling the bacterial spore contents with a long-lifetime lumophore and detecting the luminescence after a waiting period. Unattended monitoring of bacterial spores in the air comprises the steps of collecting bacterial spores carried in the air and repeatedly performing the Tb-DPA detection steps above.
Exciting the combined terbium ions and DPA generates a luminescence characteristic of the combined terbium ions and DPA. This is achieved by radiating the combined terbium ions and DPA with ultraviolet light.
U.S. Patent Application Publication No. 2004-0014154 further discloses a method for live/dead assay for bacterial spores comprising the steps of: providing a solution including terbium ions in a sample of live and dead bacterial spores; releasing DPA from viable bacterial spores by germination from a first unit of the sample; combining the terbium ions with DPA in solution released from viable bacterial spores; exciting the combined terbium ions and DPA released from viable bacterial spores to generate a first luminescence characteristic of the combined terbium ions and DPA to detect the viable bacterial spores; releasing DPA from dead bacterial spores in a second unit of the sample by autoclaving, sonication or microwaving; combining the terbium ions with the DPA in solution released from dead bacterial spores; exciting the combined terbium ions and DPA released from dead bacterial spores to generate a second luminescence characteristic of the combined terbium ions and DPA to detect the dead bacterial spores; generating a ratio of the first to second luminescence to yield a fraction of bacterial spores which are alive.
U.S. Patent Application Publication No. 2004-0014154 also discloses a method for unattended monitoring of bacterial spores in the air comprising the steps of collecting bacterial spores carried in the air; suspending the collected bacterial spores in a solution including terbium ions; releasing DPA from the bacterial spores; combining the terbium ions with DPA in solution; exciting the combined terbium ions and DPA to generate a luminescence characteristic of the combined terbium ions and DPA; detecting the luminescence to determine the presence of the bacterial spores; and generating an alarm signal when the presence of bacterial spores is detected or the concentration thereof reaches a predetermined magnitude.
Currently, bioburden levels are determined using the culture-depended methods, with which bacterial spores are quantified in terms of colony forming units (CFU's) that become visible on growth plates after incubation. There are several limitations for culture-dependent methods. First, this process requires 3-5 days to complete. Second, a large number of bacterial spores can aggregate on individual particulates giving rise to a single CFU, and thus a large underestimation of the bioburden. Third, colony-counting methods only account for cultivable spore-forming species, which constitute less than 1% in environmental samples.
It is desirable to provide a more efficient and sensitive method and apparatus for transferring all spore-forming bacteria (most especially bacteria of the genus Bacillus and Clostridium) originating on a surface, in the air or in water to a test surface, quantifying the spores, and further characterizing these spores as viable or nonviable.